Project Summary/Abstract ADP-ribose Transferase Diphtheria-like proteins (ARTDs) transfer the ADP-ribose (ADPr) moiety of nictonamide adenine dinucleotide (NAD+) onto target proteins in a highly regulated dynamic process. Curiously, this family of proteins is critical to host cell responses to different kinds of stress, including viral infection. In recent years there have been developing suggestions that ARTDs may effect an antiviral program by modulating the functionality of specific proteins during infection. Notably, a number of single stranded positive sense RNA virus families encode catalytically active macro domains, the negative regulators of ADP- ribosylation, and previous studies have shown that inactivation of these viral macro domains attenuates these viruses, reducing pathogenesis. How this is achieved at the molecular level, however, has thus far remained unclear. Notably, the complex nature of ADP-ribosylation complicates the study of macro domains with some domains acting exclusively on either monomeric (MAR) or polymeric (PAR) forms of ADPr. And despite numerous studies exploring total macro domain catalytic activity in alpha and coronaviruses, the literature regarding the role of viral macro domain PAR binding/removal is conflicting and remains underexplored. Given what is known in the literature, we hypothesize that: (i) PAR binding is specifically critical for alphavirus macro domain function, (ii) host cells up-regulate ADP-ribosylation of host proteins to inhibit infection, and (iii) viral macro domains promote virulence by counteracting ARTD mediated antiviral signaling. To address these issues, we have decided to use a combinatorial approach and employed mutagenesis and mass spectrometry in the context of Sindbis virus (SINV), a model alphavirus that encodes a macro domain, to determine the functionality of PAR binding as well as the specific ARTDs and ADP-ribosylated proteins implicated in antiviral activity. We designed mutants to abrogate PAR binding while maintaining catalytic activity to determine if these biochemical properties were critical for macro domain function. In a complementary approach, we aim to determine the specific ARTDs that are important for suppressing SINV infection, and the specific modified proteins that interact with the SINV macro domain. This builds on previous studies that investigated the in vivo implications of macro domain catalytic deficiency and moves to dissect the contributions of specific ADPr moieties, i.e. MAR or PAR. We will assess our macro domain mutants in the context of infection using defects in viral spread and innate immune signaling as markers for functional impact in various cell types, and plan to determine the specific innate immune signaling pathways that trigger or elicit ADP-ribosylation stimulation. Collectively, these experiments begin to define a post-translational mechanism employed by host cells to effect an antiviral response, dissecting the specific components that link viral macro domains to host innate immunity during infection.